Natural and Engineering Sciences 78 Volume 1, No. 3, 78-85, 2016 -RESEARCH ARTICLE- The first record of the longjaw squirrelfish, Holocentrus adscensionis (Osbeck, 1765) (Holocentriformes: Holocentridae), in the Mediterranean Sea Adriana Vella*, Noel Vella, Sandra Agius Darmanin Conservation Biology Research Group, Department of Biology, University of Malta, Msida MSD2080, Malta Abstract Research and monitoring of coastal habitats and biodiversity is increasingly contributing to the discovery of new alien species, which highlights the use of long-term monitoring for timely assessment and management due to marine environmental changes. Research work undertaken through coastal snorkelling surveys and working with fishermen allow for additional monitoring effort to record biodiversity changes and new alien species presence. One such new alien fish species was observed during spring snorkelling research while a specimen was collected in August 2016 to undertake detailed morphological, meristic and genetic analyses. Mitochondrial DNA barcoding analyses were undertaken to confirm the species' identity as the family Holocentridae is composed of a number of species that have very similar and overlapping morphological characters which may lead to misidentification. This research led to the identification of the first record of a specimen of Longjaw squirrelfish, Holocentrus adscensionis (Osbeck, 1765), in the Mediterranean Sea. This is a subtropical reef-associated species native to the Atlantic Ocean and may compete with native Mediterranean reef species. Keywords: Holocentrus adscensionis, alien, morphometrics, barcoding, Mediterranean Sea Article history: Received 20 December 2016, Accepted 23 December 2016, Available online 26 December 2016 Introduction Holocentridae is a family composed of over 84 soldier fishes and squirrelfishes, most of which are primarily nocturnal carnivorous reef-dwelling fishes which inhabit mainly tropical * Corresponding Author: Adriana Vella, e-mail: [email protected] Natural and Engineering Sciences 79 and temperate habitats (Nelson, 2006; Froese & Pauly 2016). This family is not native to the Mediterranean Sea, however specimens of genus Sargocentron have been collected and analysed from various regions in the Eastern Mediterranean. The first record of this genus in the Mediterranean Sea was along the Palestine coast and was reported in Haas and Steinitz (1947), and by 2009 it became one of the commonest fish landed in Lebanon with a 3% contribution to the total landings by artisanal Lebanese fishermen as it is commonly caught on gill-nets and long-lines (Carpentieri et al., 2009). Despite all Mediterranean records point to S. rubrum, recent genetic evidence shows that specimens from Turkey match closely to sequences of S. rubrum (Keskin and Atar, 2013), however specimens from Lebanon diverge from any currently genetically barcoded Sargocentron species (Bariche et al., 2015). This indicates that possibly more than one species of Sargocentron have entered the Mediterranean Sea (Bariche et al., 2015), however prior to this study there have been no records of Holocentrus adscensionis or any other species from the genus Holocentrus in the Mediterranean Sea. Holocentrus adscensionis is a subtropical reef-associated species native to the western Atlantic Ocean from North Carolina (USA) and the islands of Bermuda down to Brazil, including the Gulf of Mexico and several islands in the region; the mid-Atlantic ridge at St. Paul's Rocks, Ascension Island and St. Helena; and the eastern Atlantic Ocean from Gabon to Angola, including Sao Tome Island (Carpenter, 2002; Nelson, 2006; Froese & Pauly 2016). This squirrelfish is known to have a prolonged pelagic period, which includes pelagic eggs, followed by pelagic larvae and 'rhynchichthys' pelagic stage, having a total mean of 48 days (43 days to 56 days). The 'rhynchichthys' stage is then followed a 'meeki' stage which can either transform quickly to a benthic juvenile or extends its pelagic duration by an average of an additional 13 days (Tyler et al., 1993), allowing ample time for dispersal prior to settlement. Methods Snorkelling surveys along the Maltese coast, side by side with research with fishermen, allow for useful long-term monitoring of coastal biodiversity. A new alien fish species was observed during snorkelling field research work in May 2016, followed by the collection of a specimen on the 24th of August, photo of which is supplied in Figure 1. This specimen was collected from shallow coastal waters in Marsascala, Malta [GPS: 35°52'5.56"N, 14°34'29.28"E] (Figure 2). Once captured the specimen was kept for scientific analyses. The diagnostic features used in its identification followed Carpenter, 2002; Nelson, 2006; Froese & Pauly, 2016. All body length measurements were taken to the nearest 0.1 mm using electronic calipers and mass was recorded to the nearest 0.01 g. A tissue biopsy was taken and preserved in 95% ethanol. The specimen was subsequently deposited in the ichthyological collection of the Conservation Biology Research Group laboratory at the University of Malta with reference code number CBRG/F.160824/HA001. For genetic analyses, the tissue was digested with Proteinase K and the total DNA was extracted using GF-1 Tissue DNA Extraction Kit (Vivantis Technologies). PCR amplifications were carried out for: cytochrome c oxidase I gene (COI), cytochrome b (Cyt b) and 16S rRNA (16S) using primers sets as described in Vella et al., (2016a) and the 12S rRNA gene (12S) using H1478 and L1091 primers (Kocher et al., 1989). PCR amplifications were carried out following the amplification protocols described in Vella et al., (2016b). PCR products were purified and sequenced via ABI3730XL sequencer using both Natural and Engineering Sciences 80 the forward and reverse primers. The sequences, at both nucleotide and amino acid level, were analysed using Geneious v6 (http://www.geneious.com, Kearse et al., 2012). The sequences obtained were deposited in GenBank, under accession numbers KX961690-3 for COI, Cyt b, 12S and 16S respectively. These sequences were compared to other sequences available in genomic databases using BLASTn. Various COI gene sequences from different Holocentridae species available in GenBank and BOLD allowed for phylogenetic analyses of this specimen, and for comparative analyses of the current specimen to the Sargocentron haplotypes from the Mediterranean Sea. The sequences chosen were aligned using Geneious v6 (http://www.geneious.com, Kearse et al. 2012), and were trimmed to the size of the smallest homologous sequence. The genetic divergences between the 575 bp COI sequences selected were calculated using the Kimura 2- parameter distance model (Kimura, 1980), while the phylogenetic tree was constructed using 1500 bootstraps with Maximum Likelihood utilizing MEGA v6 (Tamura, 2013). Results Morphometrics The specimen caught weighs 310.82 grams and has a total body length of 301.0 mm. Its appearance, morphology and meristic counts (Table 1 and Figure 1) match the descriptions of H. adscensionis given by Carpenter, 2002; Nelson, 2006; Froese & Pauly 2016. The specimen has a moderately compressed oblong body and a long slender caudal peduncle, while the head has two very conspicuous strong spines on the sides. The anterior dorsal fin soft rays and upper caudal fin rays are elongated. The back and the upper sides of the body are reddish with some golden reflections and yellowish stripes on its back. Lighter coloured stripes get broader on the lower scale rows. The lower sides and belly are white. The snout and top of head are red, with a white streak running diagonally across the cheek. The dorsal fin spines and the interspinal membranes adjacent to the spines are yellowish-green, while the rest of the dorsal fin has a reddish pink colour. The first three anal fin spines are white while the fourth spine and the anal fin soft rays have a pink colouration (Figure 1). Figure 1. The Holocentrus adscensionis specimen caught from Marsascala, Malta on the 24th August, 2016. (Photo of the specimen on a 1x1cm scaled background). Natural and Engineering Sciences 81 Table 1. Measurements and meristic counts of the Holocentrus adscensionis specimen caught in Maltese waters. Parameter Measurements Proportion Total length 301.0 mm Fork length 238.9 mm Standard length 224.0 mm Maximum body depth 79.2 mm 35.4 % (SL) Length of dorsal fin base 127.1 mm 56.7 % (SL) Pectoral fin base 11.4 mm 5.1 % (SL) Anal fin base 32.1 mm 14.3 % (SL) Depth of caudal peduncle 17.4 mm 7.8 % (SL) Pre-pelvic length 74.0 mm 33.0 % (SL) Pre-anal length 156.0 mm 69.6 % (SL) Head length 70.3 mm 31.4 % (SL) Eye diameter 22.6 mm 10.1 % (SL) Meristic Counts Dorsal fin spines 11 Dorsal fin soft rays 15 Pelvic fin spines 1 Pelvic fin soft rays 7 Anal fin spines 4 Anal fin soft rays 10 Pectoral fin soft rays 15 Lateral line scales 50 Gill rakers (total) 24 Gill rakers on the lower limb of first gill arch 18 Genetic analyses The sequence lengths obtained were 655 bp, 792 bp, 387 bp and 578 bp for COI, Cyt b, 12S and 16S respectively. Each sequence was run via BLASTn to identify matches in GenBank. The COI sequence of the currently analysed specimen, did not match the sequences of S. rubrum from Turkey (gb:KC501233-52 - Keskin and Atar, 2013) and Sargocentron sp. from Lebanon (gb:KR861551 - Bariche et al., 2015), but rather it matched between 99% to 100% with a number of GenBank sequences belonging to H. adscensionis (Figure 3). This result matched the morphological analyses and thus confirmed the current specimen as the first record of H. adscensionis in the Mediterranean. Genetic data from the Cyt b also matches completely with the most common haplotype for H. adscensionis (DQ380026) as recorded in various regions across the Atlantic Ocean (Bowen et al., 2006). Data on the 12S and 16S genes of H. adscensionis is absent and thus it was not possible to use these genes for identification down to the species level although they confirmed the genus by matching with H.